The present invention relates to microorganisms for preventing or treating obesity or diabetes mellitus, which are capable of reducing an amount of monosaccharides or disaccharides that can be absorbed into the intestine by converting those mono or disaccharides into polymeric materials that cannot be absorbed in the intestines. The present invention also relates to use of the microorganisms for preventing or treating obesity or diabetes mellitus and a pharmaceutical composition containing the microorganisms.
Obesity is well known as a chronic disease caused by various factors whose origins have not yet been clearly discovered. It is understood that obesity induces hypertension, diabetes mellitus, coronary heart disease, gall bladder disease, osteoarthritis, sleep apnea, respiratory disorder, endomerial, prostate, breast and colon cancer and the like.
According to the NIH Report (THE EVIDENCE REPORT: Clinical Guideline on the Identification, Evaluation, and Treatment of Overweight and Obesity in Adults, 1999, NIH), about 97,000,000 Americans suffer from overweighting and obesesity, and the number of patients of type II diabetes mellitus associated with obesity, reaches about 15,700,000. Moreover, it is reported that about 200,000 people die of diseases associated with obesity each year (Dan Ferber, Science, 283, pp 1424, 1999).
Diabetes mellitus is one of the most widespread chronic diseases in the world, which impose a substantial expense on the public as well as on patients of diabetes mellitus and their families.
There are several types of diabetes mellitus that are caused by various etiological factors and whose pathogenesis is different from each other. For example, genuine diabetes mellitus is characterized by high level of blood glucose and glycosuria, and is a chronic disorder of carbohydrate metabolism due to a disturbance of the normal insuline mechanism.
Non-Insulin-Dependent Genuine Diabetes Mellitus (NIDDM), or the type II diabetes mellitus is found in adults who have insulin-resistance in a peripheral target tissue, despite of normal generation and function of insulin. Non-Insulin-Dependent Genuine Diabetes Mellitus(NIDDM) can be caused by three important metabolic disorders, i.e., insulin-resistance, fucntional disorder of insulin secretion stimulated by nutrients, and overproduction of glucose in liver. Failure to treat NIDDM, resulting in losing control of blood glucose levels, leads to death of patients from diseases such as atherosclerosis, and/or may cause late complications of diabetes, such as retinopathy, nephropathy or neuropathy.
Accompanying diet-exercise therapy, NIDDM therapy uses sulfonylurea and biguanidine compounds to control blood glucose levels. Recently, therapeutic compounds such as metformin or acarbose have been used for treating NIDDM. However, diet-exercise therapy alone or even combined with chemotherapy using such compounds fails to control hyperglycemia in some of the diabetes mellitus patients. In such cases, these patients require exogenous insulin.
Administration of insulin is very expensive and painful to patients, and furthermore, may cause various detrimental results and various complications in patients. For example, incidences, such as, miscalculating insulin dosage, going without a meal or irregular exercise, may cause insulin response (hypoglycemia) and sometimes the insulin response occurs even without any particular reasons. Insulin injection may also cause an allergy or immunological resistance to insulin.
There are several methods for preventing or treating obesity or diabetes mellitus, including diet-exercise therapy, surgical operation and chemotherapy. Diet-exercise therapy involves a low-calorie and low-fat diet accompanying aerobic exercise, but this therapy requiring a regular performance is hard to continue until achieving the goal.
Despite of instant effects, a surgery for physically removing body fat has limitations due to the risk and cost involved in a surgical operation and insufficient durability of the effects.
As one of the most promising therapies currently developed, pharmacotherapy can reduce blood glucose level, inhibit absorption of glucose, strengthen the action of insulin or induce the decrease of appetite. The medicines that have been developed so far use various physiological mechanisms for the prevention and the treatment of obesity and diabetes mellitus.
Some medicines, such as, sulfonylurea, metformin, pioglitazone or thiazolidindione derivatives and the like have been developed to enhance the function of insulin. Although sulfonylurea stimulates insulin-secretion from xcex2-cells in the pancreas, it may accompany side effects, such as hypoglycemia resulting from lowering blood glucose levels under normal levels.
Metformin is mainly used for insulin-nondependent diabetes mellitus patients who fail to recover after diet-exercise therapy. This medicine inhibits hepatic gluconeogenesis and enhances glucose disposal in muscle and adipose tissue. However, it suffers from side effects, such as, nausea, vomiting and diarrhea.
Pioglitazone developed by Takeda in Japan, enhances the function of insulin through increasing susceptibility of cells to insulin (Kobayashi M. et al., Diabetes, 41(4), pp 476-483, 1992).
Beta 3-adreno receptor inhibitor (BRL-35135) known as a medicine that stimulates the decomposition of body fats and that convert body fats into heat with a specific action on adipose cells, also suffers from lowerings blood glucose level.
The inhibitor of a pancreatic lipase (Orlistat produced by Roche of Switzlend) inhibits and/or reduces absorption of body fats by inhibiting pancreatic lipase. It, however, accompanies undersirable inhibition of absorption of fat-soluble vitamin and may also cause breast cancer.
Generally, medicines that decrease appetite affects catecholamine in the brain. However, dexfenfluororamine and fenfluoroamine have side effects of nerve toxicity and valvular heart disease. Also, sibutramine has side effects of increasing heart rate and blood pressure.
xcex1-Glucosidase inhibitor (Acarbose produced by Bayer of Germany), is known as a glucose absorbing inhibitor. Acarbose is pseudo-monosaccharide which competitively inhibits the action of various a-glucosidases existing in microvilli of the gastrointestinal tract. However, taking a large amount of these may induce diarrhea. (W. Puls et al., Front. Horm. Res. 2, 235, 1998).
Amylase inhibitor that inhibit converting carbohydrates into oligosaccharides has been developed to prevent imbalance of metabolism originated from excessive uptake of nutrient. (Sanches-Monge R. et al. Eur. J. Biochem., 183, 0037-40, 1989).
Dietary fiber using diet with a large amount of vegetable fiber is the easiest way to obtain inhibitory effect on obesity by lowering glucose and/or fat amounts absorbed in the intestine. However, such method also involves problems in requiring facility and manpower for the production of dietary fiber with low productivity.
Polymeric materials, such as, isomaltotriose, dextran and pullulan, inhibit the increase of blood glucose level originated from glucose. However, such materials also cause severe side effects. For example, dextran may induce excessive bleeding by delaying a blood coagulation time.
Among said various medicines, dietary fibers are the most useful medicine for prevention or treatment of obesity because no damage to the human metabolism-balance and use natural substances.
Microorganism dietary fiber is produced using microorganisms, such as, Gluconobacter sp., Agrobacterium sp., Acetobacter xylinum, A. hansenil, A. pasteurianus, A. aceti, Rhizobium sp., Alcaligenes sp., Sarcina sp., Streptococcus thermophilus, Lactococcus cremoris, Lactobacillus helveticus, Lactobacillus bulgaricus, Lactobacillus sake, Lactobacillus reuteri, Lactobacillus lactis, Lactobacillus delbrueckii subsp., Lactobacillus helveticusglucose var. jugurti, Leuconostoc dextranicum, Bulgariscus sp., Campestris sp., Sphingomonas sp.
Dietary fiber produced by these microorganisms is used as stabilizer, thickening agent, emulsifier, hygroscopic agent of various foods and raw materials of cosmetics and pharmaceuticals. Microorganism cellulose, xanthan, acetan, guar gum, locust bean gum, carrageenan, alginate, and agar obtained from seaweed are commercialized.
Lactobacillus sp. strain is the major component of normal microbial flora in the human intestines. Its significant roles for maintaining digestive organ and for healthy environment of the vagina, have been well known. [Bible, D. J., ASM News, 54:661-665, 1988; Reid G. and A. W. Bruce, In H Lappin-Scott (de.), Bacterial biofilms, Cambridge University Press, Cambridge, England, p. 274-281, 1995; Reid G., A. W. Bruce, J. A. McGroarty, K. J. Cheng, and J. W. Costerton, clin. Microbiol. Rev., 3:335-344, 1990]. Generally, Lactobacillus strain inhabits in digestive organs (L. acidophilus, L. intestinalis, L. johnsonii, L. reuteri et al.,), muscosa of the vagina (L. vanginals, L. gasseri), food (wine-L. hilgardii), lactobacillus beverage (L. kefir, L. kefiranofaciens), cheese (L. casey), vinegar (L. acetotolerance), the oral cavity (L. oris), yeast (L. sake, L. homohiochi), fruit juice (L. kunkeei, L. mali, L. suebicus), fermented sausages or fish (L. farciminis, L. alimentarious) et al.
Many people take health complementary food containing a Lactobacillus sp. strain in order to maintain healthy intestines and to prevent urogenital tract infection. Recently, in addition to the prevention of the diarrhea, constipation and urogenital tract infection; various probiotic activities of Lactobacillus, such as, control of immunity, control of cholesterol level in blood, prevention of cancer, treatment of rheumatism, alleviation of sensitivity on lactose or effect for atopic dermatitis, have been reported and thus, have attracted more attention.
According to the U.S. Public Health Service Guideline, all of the 262 Lactobacillus deposited in ATCC are classified as xe2x80x9cBio-safety Level 1,xe2x80x9d which stands for no potential risk, which has been known up to now, causing diseases in human or animals. There is no harm to human body among approximately 60 strains of Lactobacillus. 
Recently, there has been a rapid progress in the research for an extracellular dietary fiber produced by Lactobacillus. It has been reported that a process of producing dietary fiber in these strains are very complicated because a lot of genes are mediated in the process, and the amount of dietary fiber thus produced are very low (Int. J. Food Microbiol., Mar 3 40:1-2, 87-92, 1998; Current Opinion in Microbiology, 2:598-603, 1999; Appl. Environ. Microbiol., Feb 64:2, 659-64, 1998; FEMS Microbiol. Rev. Apr 23:2 153-77, 1999; FEMS Microbiol. Rev. Sep 7:1-2, 113-30, 1990).
Also, various researches on the synthesis of cellulose by Acetobacter sp. which is well known as a microorganism producing dietary fiber, have been performed (Aloni Y., cohen R., Benziman M., Delmer D, J Biological chemistry, 171:6649-6655, 1989; Ascher M., J. Bacteriology, 33:249-252, 1937; Benziman M., Burger-Rachamimv H., J., Bacteriology, 84:625-630, 1962; Brown AM. Journal of Polymer science, 59:155-169, 1962; Brown AM, Gascoigne JA, Nature, 187:1010-1012, 1960; Calvin JR, Planta DP, Benziman M., Padan E, PANS USA, 79:5282-5286, 1982; Dehmer DP. Brown RM Jr., Cooper JB, Lin FC, Science, 230:82-825, 1985).
Acetobacter is a strict aerobe but has characteristics of surviving and living under the condition of infinitesimal oxygen, and of being floated to seek for oxygen by means of synthesizing cellulose dietary fiber itself under this condition of infinitesimal oxygen. According to the research regarding the amount and rate of converting glucose into cellulose dietary fiber by Acetobacter (Brown et al.: Proc. Natl. Acad. Sci. USA, Vol73 (12), 4565-4569), Acetobacter converts glucose into cellulose with the speed rate of 400 mol/cell/hour. This is equivalent to the rate that about 200 g glucose can be converted into cellulose dietary fiber by 4xc3x971015 cells per an hour.
Although Acetobacter that can metabolizes saccharose is rare, Acetobacter converting sacchores in glucose, exists in nature (PNAS, 9: pp14-18). Presently, FDA of the United States has approved Acetobacter xylinum for synthesizing acetic acid and sorbose, and has classified it as generally safe microorganism (GRAS: Generally Recognized As Safe).
As mentioned above, although there have been various researches and efforts to develop drugs for treatment or prevention of obesity and diabetes mellitus, their results were not satisfactory. Various chemical substances mentioned above, have been developed for treatment of obesity and diabetes mellitus, but still suffer from various side effects. These drugs forcibly discharge body fat together with valuable proteins. Consequently, any one single drug for treatment or prevention of obesity and diabetes mellitus at the origin thereof does not exist yet.
Therefore, the object of the present invention is to provide microorganisms capable of living within the intestines and converting oligosaccharides produced by the digestive enzymes into non-digestable polysaccharides, and thereby remarkably reducing the amount of oligosaccharide absorbed into the intestines.
Another object of the present invention is to provide a pharmaceutical composition comprising at least one of said microorganisms in an amount effective to prevent or treat obesity and diabetes mellitus and a pharmaceutically acceptable carrier. Another object of the present invention is to provide a method for preventing or treating obesity, diabetes mellitus comprising administering to a subject in need thereof capable of pharmaceutical comprising a method for reducing weight gain, controlling blood glucose level and control absorption of blood lipod.
The microorganisms that can be used for the pharmaceutical composition of the present invention preferably fall within Acetobacter genus, Gluconobacter genus, Lactobacillus genus, and Acrobacterium genus, which are capable of living in the intestine and not harmful to human body, and are capable of converting oligosaccharides into polysaccharides that cannot be absorbed into human body. Specifically, the following microorganisms can be used as microorganisms of the pharmaceutical composition of the present invention, such as, Acetobacter xylinum, A. hansenii, A. pasteurianus, A. aceti, Lactococcus cremoris, Lactobacillus helveticus, L. bulgaricus, L. sake, L. reutari, L. lactis, the subspecies of L. delbrueckii, L. delbrueckii subsp., and a variant form of L. helveticusglucose. Preferably, the microorganisms can be used as an active principle of the pharmaceutical composition of the present invention is Lactobacillus sp. BC-Y009 (KCTC0774BP) strain or Acetobacter sp. BC-Y058 (KCTC0773BP) strain.
The pharmaceutical composition of the present invention may be administered in a form of tablet, capsule, suspension or emulsion, which comprises excipients, pharmaceutically acceptable vehicles and carriers which are selected depending on administration routes. The pharmaceutical formulation of the present invention may further comprises supplemental active ingredients.
Lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginic acid salt, treguhkense latex, gelatin, calcium silicate, finecrystalline cellulose, polyvinylpyrolidon, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate and prophylhydroxybenzoate, talc, magnesium stearate or mineral oil may be used as carriers, exipients or diluents in the pharmaceutical composition of the present invention.
In addition, the pharmaceutical composition of the present invention may further comprises lubricants, moisturizer, emulsifier, suspension stabilizer, preservative, sweetener and flavor. The pharmaceutical composition of the present invention may be in a form of an enteric coating formulation produced by various methods which have been publicly known, in order to deliver the active ingredients of the pharmaceutical composition, ie., microorganisms, to the small intestines without degradation by gastric juices in stomach.
Furthermore, microorganisms of the present invention may be administered in a form of capsule prepared by conventional process. For example, standard vehicles and lyophilized microorganisms of the present invention are mixed together and prepared to pellets and then, the pellets are filled into hard gelatin capsules. In addition, the microorganisms of the present invention and pharmaceutically allowable vehicles, for example, aqueous gum, cellulose, silicate or oil are mixed to produce a suspension or emulsion and then, this suspension or emulsion may be filled into soft gelatin capsule.
The pharmaceutical composition of the present invention may be prepared as an enterically coated tablets or capsules for oral administration. The term xe2x80x9cthe enteric coatingxe2x80x9d of this application includes all conventional pharmaceutically acceptable coating that has resistance to gastric juice, however, in the small intestines, can disintegrate sufficiently for a rapid release of the microorganisms of the present invention.
The enteric coating of the present invention can be maintained for more than 2 hours in synthetic gastric juice, such as HCl solution of pH 1 at the temperature of 36xc2x0 C. to 38xc2x0 C. and desirably, decomposes within 0.5 hours in synthetic intestinal juice, such as KH2PO4 buffer solution of pH 6.8.
The enteric coating of the present invention applies to each tablet with the amount of about 16 to 30 mg, desirably 16 to 25 mg, more desirably 16 to 20 mg. The thickness of enteric coating of the present invention is 5 to 100 xcexcm, desirably 20 to 80 xcexcm. The components of the enteric coating are selected appropriately from common polymeric materials which have been publicly well known. The polymeric materials which may be employed for enteric coating of the present invention are enumerated and described in the flowing articles [The Theory and Practices of Industrial Pharmacy, 3rd Edition, 1986, pp. 365-373 by L. Lachman, Pharmazeutische Technologie, thieme, 1991, pp. 355-359 by H. Sucker, Hagers Handbuch der Pharmazeutischen Praxis, 4th Edition, Vol. 7, pp. 739, 742, 766, and 778, (SpringerVerlag, 1971), and Remington""s Pharmaceutical Sciences, 13th Edition, pp. 1689 and 1691 (Mack Publ., Co., 1970)]. For example, cellulose ester derivative, cellulose ether and copolymer of acryl and methyl acrylate or maleic acid or phthalic acid derivative may be used in enteric coating of the present invention.
The preferred enteric coating of the present invention are prepared from polymers of cellulose acetate phthals or trimelitate and methacrylic copolymer (for example, copolymer of more than 40% of methacrylic acid and methacrylic acid which contains hydroxyprophyl methylcellulose phthalate or derivatives from ester thereof).
Endragit L 100-55 manufactured by Rohm GmbH of Germany may be used as a raw material for the enteric coating of the present invention.
Cellulose acetate phthalate employed in the enteric coating of the present invention, has about 45 to 90 cP of viscosity, 17 to 26% of acetyl contents and 30 to 40% of phthalate contents. The cellulose acetate trimelitate used in the enteric coating, has about 15 to 21 cS of viscosity, 17 to 26% of acetyl contents, and 25 to 35% of trimelityl contents. The cellulose acetate trimelitate which is manufactured by the Eastman Kodak Company may be used as a material for the enteric coating of the present invention.
Hydroxyprophyl methylcellulose phthalate used in the enteric coating of the present invention has molecular weight of generally 20,000 to 100,000 dalton, desirably 80,000 to 130,000 dalton and has 5 to 10% of hydroxyprophyl contents, 18 to 24% of metoxy contents, and 21 to 35% of phthalyl contents. Cellulose acetate phthalate manufactured by the Eastman Kodak Company can be used as a material for the enteric coating of the present invention.
Hydroxyprophyl methylcellulose phthalate used in the enteric coating of the present invention is HP50 which is manufactured by the Shin-Etsu Chemical Co. Ltd., Japan. The HP50 has 6 to 10% of hydroxyprophyl contents, 20 to 24% of metoxy contents, 21 to 27% of prophyl contents, and molecular weight is 84,000 dalton. Another material for enteric coating manufactured by the Shin-Etsu Chemical Co. Ltd., is HP55. HP55 can also be used as material for the enteric coating of the present invention. The HP55 has 5 to 9% of hydroxyprophyl contents, 18 to 22% of metoxy contents, 27 to 35% of phthalate contents, and molecular weight is 78,000 dalton.
The enteric coating of the present invention is prepared by using conventional methods of spraying the enteric coating solution to the core. Solvents used in the process of the enteric coating are alcohol such as ethanol, ketone such as acetone, halogenated hydrocarbon such as dichloromethane, or the mixture thereof. Softeners such as Di-n-butylphthalate and triacetin are added to the enteric coating solution in the ratio of 1 part coating material to about 0.05 or to about 0.3 part softner.
A spraying process is preferably performed continuously, and the amount of materials sprayed may be controlled depending on the condition of the coating process. Spraying pressure may be regulated variously and, generally, desirable result can be obtained under the pressure of average 1 to 1.5 bar.
xe2x80x9cThe effective amountxe2x80x9d of this specification means the minimum amount of the microorganisms of the present invention, which can reduce the amount of oligosaccharide absorbed into the body through the intestines of mammalian animals. The amount of microorganisms administered into a body with the pharmaceutical composition of the present invention may be adjusted depending on the administration method and the administration subject.
The composition of the present invention may be administered once or more per day on the subject. The unit of administration amount means that it is separated physically and thus is suitable for the unit administration for the human subjects and all other mammalian animals. Each unit contains a pharmaceutically acceptable carrier and the amount of the microorganisms of the present invention which are effective in therapy.
An oral administration unit of an adult patient contains microorganisms of the present invention in an amount, desirably, 0.1 g or more, and the composition of the present invention contains 0.1 to 10 g per one time administration, desirably 0.5 to 5 g. The effective amount of microorganisms of the present invention is 0.1 g per 1 day.
However, the administration amount can vary depending on the weight and the severity of obesity of the patient, supplemental active ingredients included and microorganisms used therein. In addition, it is possible to divide up the daily administration amount and to administer continuously, if needed. Therefore, range of the administration amount does not limit the scope of the present invention in any way.
The xe2x80x9ccompositionxe2x80x9d of the present invention means not only as medicinal products but also to serve as functional foods and health complementary foods.
In case of taking the composition of the present invention periodically, microorganisms form colony within the intestines and interrupt absorption of oligosaccharide in the body competitively. Also, non-digestable fibers produced by microorganisms make a healthy condition for other useful microorganisms within the intestines and stimulate the intestinal activity. Consequently, the composition of the present invention functions to treat and prevent obesity and diabetes mellitus.